Seasoning products with high content of sweet amino acids and yeast strain for use in obtaining the same

ABSTRACT

The present invention provides a seasoning serving as an alternative to hydrolyzed animal proteins. 
     The present invention provides a seasoning composition comprising free proline in an amount of 8.0% or more of the free amino acid composition, wherein the seasoning composition is not derived from collagen or gelatin; a seasoning composition comprising free proline in an amount of 6.0% or more of the free amino acid composition and free glycine in an amount of 5.0% or more of the free amino acid composition, wherein the seasoning composition is not derived from collagen or gelatin; a yeast extract comprising free proline in an amount of 8.0% or more of the free amino acid composition; and a seasoning composition comprising a yeast extract, wherein the seasoning composition comprises free proline in an amount of 6.0% or more of the free amino acid composition and free glycine in an amount of 5.0% or more of the free amino acid composition, etc. The present invention also provides a yeast strain for use in preparing such a seasoning composition and such a yeast extract.

TECHNICAL FIELD

The present invention relates to a seasoning, particularly a yeastextract. The seasoning of the present invention is useful in the fieldof food industry.

BACKGROUND ART

Sweetness, a taste typical of sugar, is one of the five primary tastes,and saccharides such as sugar and glucose are primarily used as naturalsweeteners. Also in the case of amino acids, proline, glycine andalanine are known to be sweet and may also be used for enhancement ofsweetness, as circumstances demand, because the sweetness quality ofthese amino acids is different from that of saccharides.

Likewise, hydrolyzed animal proteins used frequently as seasonings inprocessed foods may also be used for enhancement of sweetness, becausesuch hydrolysates mainly originate from collagen or gelatin, and hencehave a characteristic sweetness rich in proline, glycine and alaninewhich are constituent amino acids of collagen or gelatin.

Yeast extracts are also used as seasonings in various foods (PatentDocuments 1 to 3). For the application of yeast mutant strains in thefield of food industry, some reports have been issued. For example,there is a report showing that mutant strains tolerant to AZC (a toxicanalog of proline) have been screened to obtain a mutant strain whichallows intracellular proline accumulation and is more tolerant tofreezing stress than its parent strain (Non-patent Document 1).According to the same procedures or the like, a freeze-tolerant yeaststrain which allows intracellular accumulation of one or more aminoacids selected from proline, arginine, lysine and glutamic acid has beenused in frozen bread dough (Patent Document 4). Likewise, aproline-accumulating yeast transformant has been used for brewing ofJapanese rice wine (Patent Document 5). However, the former reportfocuses on the survival rate of yeast in frozen bread dough, while thelatter report is directed to the ethanol tolerance of yeast and/ormodification of Japanese rice wine to have a milder taste. Neither ofthem provides any information about the use of yeast as a seasoning.Moreover, neither of them aims to enhance a hydrolyzed animalprotein-like sweetness or provides any information about the overallsweetness of proline, alanine and glycine.

Patent Document 1: JP 2005-102549 A

Patent Document 2: JP 10-327802 A

Patent Document 3: WO99/16860

Patent Document 4: JP 9-234058 A

Patent Document 5: JP 2006-67806 A

Non-patent Document 1: H. Takagi, F. Iwamoto, and S. Nakamori, Appln.Microbiol. Biotecnol, 47, 405-411, 1977

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

Since hydrolyzed animal proteins are prepared through hydrochloric acidhydrolysis, it is known that substances suspected to be toxic, which arecalled monochloropropanediol (MCP) and dichloropropanol (DCP), aregenerated when the preparation is accomplished in a standard manner.Preparation techniques have been developed to give safe seasonings byreducing MCP and DCP, so that recent products have low MCP and DCPcontents. However, there is still a great demand for alternativeseasonings.

On the other hand, yeast extracts are derived from yeast cells and henceare rich in non-synthetic amino acids. Yeast cells are particularly richin glutamic acid (a flavor component) and alanine (a sweet component) asfree amino acids, and are expected as alternatives to hydrolyzed animalproteins. However, it has been difficult for yeast cells to provide asweetness characteristic of hydrolyzed animal proteins, because theirglycine and proline contents are particularly low.

Means for Solving the Problems

As a result of extensive and intensive efforts made to solve theproblems stated above, the inventors of the present invention have foundthat yeast strains modified to increase their proline content arescreened to find out those rich in glycine without any difference intheir alanine content, thereby making it possible to obtain yeast cellsand yeast extracts rich in these amino acids. This finding led to thecompletion of the present invention.

[Yeast Extract]

The present invention provides the following aspects.

1) A yeast extract comprising free proline in an amount of 8.0% or moreof the free amino acid composition.

2) The yeast extract according to 1) above, which comprises free prolinein an amount of 15.0% or more of the free amino acid composition.

3) A seasoning composition comprising a yeast extract, wherein theseasoning composition comprises free proline in an amount of 6.0% ormore of the free amino acid composition and free glycine in an amount of5.0% or more of the free amino acid composition.

4) A seasoning composition comprising a yeast extract, wherein theseasoning composition is supplemented with amino acids to have the aminoacid composition shown in any one of 1) to 3) above.

The present invention also provides the following aspects.

1′) A method for preparing a yeast extract comprising free proline in anamount of 8.0% or more of the free amino acid composition, whichcomprises the steps of: culturing cells of a strain belonging to thegenus Saccharomyces or Candida and having a high proline-producingcapacity and, if desired, having a high alanine-producing capacity underconditions effective for proline production (if desired, underconditions effective for proline production and also effective forglycine production) to obtain yeast cells; and obtaining the yeastextract from the resulting yeast cells.

2′) The method according to 1′ above, which is intended for preparationof a yeast extract comprising free proline in an amount of 15.0% or moreof the free amino acid composition.

3′) A method for preparing a seasoning composition comprising a yeastextract, wherein the seasoning composition comprises free proline in anamount of 6.0% or more of the free amino acid composition and freeglycine in an amount of 5.0% or more of the free amino acid composition.

4′) A method for preparing a seasoning composition comprising a yeastextract, wherein the seasoning composition is supplemented with aminoacids to have the amino acid composition shown in any one of 1′) to 3′)above.

When reference is made herein to amino acids, such amino acids do notinclude amino acid residues constituting a part of a peptide or protein,and refer to amino acids in a free form, unless otherwise specified.

As used herein, the term “yeast extract” refers to an extract obtainedfrom yeast cells by solvent extraction, unless otherwise specified.Purified, concentrated and dried forms of an extract also fall withinthe context of “yeast extract” as long as they are obtained through theextraction step and contain at least several types of amino acids.

When reference is made herein to content or composition values forcomponents (e.g., amino acids) of a yeast extract or seasoningcomposition, these values are determined on a weight basis, unlessotherwise specified.

The proline content in conventional yeast extracts is about 0.5% to 1%of the amino acid composition, whereas the proline content in the yeastextract of the present invention is 6.0% or more, preferably 8.0% ormore, more preferably 9.0% or more, more preferably 13.0% or more, morepreferably 14.0% or more, more preferably 15.0% or more, and even morepreferably 17% or more. Likewise, the glycine content in conventionalyeast extracts is about 1% to 2% of the amino acid composition, whereasthe glycine content in the yeast extract of the present invention is4.0% or more, preferably 5.0% or more, and more preferably 5.1% or more.Further, the alanine content in the yeast extract of the presentinvention is 12% to 17% of the amino acid composition, which iscomparable to that of conventional yeast extracts.

In a preferred embodiment of the yeast extract of the present invention,the proline content is 6.0% of the amino acid composition and theglycine content is 4.0% of the amino acid composition. In addition, itis preferred that the alanine content is 12% to 17% of the amino acidcomposition.

In a more preferred embodiment, the proline content is 5.0% of the aminoacid composition and the glycine content is 5.0% of the amino acidcomposition. In addition, it is preferred that the alanine content is12% to 17% of the amino acid composition.

In an even more preferred embodiment, the proline content is 6.0% of theamino acid composition and the glycine content is 5.0% of the amino acidcomposition. In addition, it is preferred that the alanine content is12% to 17% of the amino acid composition.

In another preferred embodiment, it is preferred not only that theseamino acid contents are set at or above the defined values, but alsothat the content of bitter amino acids such as leucine, isoleucine andarginine is low, for example, ⅔ or less than that of conventional yeastextracts.

As described above, since the yeast extract of the present invention isrich in sweet amino acids, i.e., proline, glycine and alanine, it is ofadvance taste type, has a clear and well-harmonized taste, and preventsany heavy flavor from remaining in the mouth, when compared toconventional yeast extracts. The yeast extract of the present inventionalso presents a sweetness close to that of hydrolyzed animal proteins.

The yeast extract of the present invention can be prepared from yeastcells obtained as follows: strains having a high proline-producingcapacity are screened to find out a strain whose alanine content iscomparable to that of conventional yeast strains and which also has ahigh glycine-producing capacity.

Strains having a high proline-producing capacity can be obtained asfollows: their parent strains are mutated by irradiation withultraviolet or radioactive rays or by treatment with an agent such asethylmethanesulfonate (EMS) or N-methyl-N′-nitro-N-nitrosoguanosine(NTG), and the resulting mutant strains are then screened to selectstrains that are tolerant to a proline analog, such as thioproline,azetidine-2-carboxylate (AZC) or dehydroproline. As to detailedprocedures for obtaining mutant strains, reference may be made to, e.g.,Non-patent Document 4 (supra) and also to the Example section describedlater.

As parent strains, various edible yeast strains may be used. Preferredfor use are yeast strains belonging to the genus Saccharomyces (e.g.,those belonging to Saccharomyces cerevisiae, Saccharomyces rosei,Saccharomyces uvarum and Saccharomyces chevalieri) or yeast strainsbelonging to the genus Candida (e.g., those belonging to Candidautilis).

For culturing these parent strains and mutant strains, it is possible touse a medium used frequently for yeast culturing, such as YPD medium ormolasses medium. If desired, it is possible to use a carbon source suchas glucose, sucrose, molasses or a saccharified solution and/or anitrogen source such as ammonium sulfate, ammonium chloride, nitratesalt, urea or ammonia. The medium may be supplemented with minerals(e.g., phosphoric acid, potassium, magnesium, zinc, copper, manganese,iron), vitamins and/or amino acids.

Once strains having a high proline-producing capacity have beenobtained, the strains thus obtained can be screened to select a strainhaving a high glycine-producing capacity and a high alanine-producingcapacity, if desired. The determination of whether the resulting strainhas high producing capacities can be accomplished by using prior arttechniques (e.g., using an amino acid analyzer) to test the resultingyeast extract. It is also possible to select a strain having not onlyhigh producing capacities for the above amino acids, but also lowproducing capacities for bitter amino acids such as leucine, isoleucineand arginine.

A particularly preferred example of yeast which can be used forobtaining the yeast extract of the present invention is Saccharomycescerevisiae JT-YE-P-52 (Accession No. ABP-10725) obtained herein in theExample section, which will be described later.

To prepare a yeast extract from yeast cells, the mutant strain obtainedas above is first cultured under conditions effective for prolineproduction (preferably, under conditions effective for prolineproduction and also effective for glycine and/or alanine production).The culture temperature may be set to 20° C. to 38° C. As a medium, theabove YPD medium, molasses medium or the like may be used. The medium pHmay be adjusted to 3.5 to 8.0. The culture period may be set to 1 to 48hours, for example, 8 to 36 hours. During culturing, it is possible tomake shaking, agitation, aeration, or addition of the medium oradditional ingredients, when required. An example of effectiveconditions is shaking or spinner culturing for 10 hours or longer usingYPD medium (1.0% Bacto yeast extract, 2.0% Bacto peptone, 2.0% glucose).

Once the cultured product has been obtained, yeast cells are obtainedfrom the cultured product through an appropriate means such ascentrifugation, and then washed (when required), followed by boilingwater extraction, enzymolysis and/or autolysis to give an extract.

Conditions for culturing and extraction may be determined as appropriateby those skilled in the art. As to detailed procedures for this purpose,reference may be made to the Example section described later.

[Seasoning Composition]

The inventors of the present invention have also found that a seasoningcomposition having the same amino acid composition as the above yeastextract is excellent, e.g., in terms of presenting a sweetness close tothat of hydrolyzed animal proteins, as in the case of the above yeastextract. Thus, the present invention also provides the followingaspects.

5) A seasoning composition comprising free proline in an amount of 8.0%or more of the free amino acid composition, wherein the seasoningcomposition is not derived from collagen or gelatin.

6) The seasoning composition according to 5) above, which comprises freeproline in an amount of 15.0% or more of the free amino acidcomposition.

7) A seasoning composition comprising free proline in an amount of 6.0%or more of the free amino acid composition and free glycine in an amountof 5.0% or more of the free amino acid composition, wherein theseasoning composition is not derived from collagen or gelatin.

The present invention also provides the following aspects.

5′) A method for preparing a seasoning composition without usingcollagen or gelatin, which comprises the step of incorporating freeproline in an amount of 8.0% or more of the free amino acid composition.

6′) The method for preparing a seasoning composition according to 5′)above, which comprises the step of incorporating free proline in anamount of 15.0% or more of the free amino acid composition.

7′) A method for preparing a seasoning composition without usingcollagen or gelatin, which comprises the step of incorporating freeproline in an amount of 6.0% or more of the free amino acid compositionand free glycine in an amount of 5.0% or more of the free amino acidcomposition.

To obtain the seasoning composition of the present invention, sourcematerials other than the animal protein collagen or gelatin are used,including yeast extract, vegetable extract, bonito extract, dried bonitoextract, kelp extract, pork meat extract, beef meat extract, chickenmeat extract, raw fish sauce, soy sauce, miso (bean paste), etc.

The proline content in seasoning compositions derived from conventionalyeast extracts is about 0.5% to 1% of the amino acid composition,whereas the proline content in the present invention is 6.0% or more,preferably 8.0% or more, more preferably 9.0% or more, more preferably13.0% or more, more preferably 14.0% or more, more preferably 15.0% ormore, and even more preferably 17% or more. Likewise, the glycinecontent in seasoning compositions derived from conventional yeastextracts is about 1% to 2% of the amino acid composition, whereas theglycine content in the present invention is 4.0% or more, preferably5.0% or more, and more preferably 5.1% or more. Further, the alaninecontent in the present invention is 12% to 17% of the amino acidcomposition, which is comparable to that of conventional yeast extracts.

In a preferred embodiment of the seasoning composition of the presentinvention, the proline content is 6.0% of the amino acid composition andthe glycine content is 4.0% of the amino acid composition. In addition,it is preferred that the alanine content is 12% to 17% of the amino acidcomposition.

In a more preferred embodiment, the proline content is 5.0% of the aminoacid composition and the glycine content is 5.0% of the amino acidcomposition. In addition, it is preferred that the alanine content is12% to 17% of the amino acid composition.

In an even more preferred embodiment, the proline content is 6.0% of theamino acid composition and the glycine content is 5.0% of the amino acidcomposition. In addition, it is preferred that the alanine content is12% to 17% of the amino acid composition.

In another preferred embodiment, it is preferred not only that theseamino acid contents are set at or above the defined values, but alsothat the content of bitter amino acids such as leucine, isoleucine andarginine is low, for example, ⅔ or less than that of conventional yeastextracts.

The seasoning composition of the present invention may be prepared byusing the above strain which has a high proline-producing capacity and,if desired, also has a high alanine- and/or glycine-producing capacity,or alternatively, may be obtained by incorporating a conventionallyprepared yeast extract with proline and, if desired, with glycine togive the defined amount(s).

[Novel Yeast]

The present invention also provides the following yeast strain for usein preparing the yeast extract of the present invention and theseasoning composition of the present invention.

8) A yeast strain belonging to the genus Saccharomyces or Candida andhaving a tolerance to azetidine-2-carboxylic acid (AZC), wherein uponculturing under conditions effective for proline production, a boilingwater extract of the resulting cells may comprise free proline in anamount of 8.0% or more of the free amino acid composition.

9) The yeast strain according to 8) above, which is Saccharomycescerevisiae JT-YE-P-52 (Accession No. ABP-10725) or a mutant strainthereof having the same microbiological properties.

The present invention also provides the following aspects.

8′) A method for screening a yeast strain, which comprises the steps of:selecting strains tolerant to a proline analog from mutant strainsderived from yeast strains belonging to the genus Saccharomyces orCandida; and selecting a strain having a high proline-producing capacityand, if desired, having a high glycine-producing capacity and/or a highalanine-producing capacity from the strains tolerant to a prolineanalog.

8″) A method for creating a yeast strain, which comprises the steps of:selecting strains tolerant to a proline analog from mutant strainsderived from yeast strains belonging to the genus Saccharomyces orCandida; and selecting a strain having a high proline-producing capacityand, if desired, having a high glycine-producing capacity and/or a highalanine-producing capacity from the strains tolerant to a prolineanalog.

As used herein, when a boiling water extract of the resulting cells maycomprise a specific free amino acid (e.g., proline) in an amount of X%or more of the free amino acid composition, it is intended to explainthe maximum capacity (proline-producing capacity) which can be achievedby a target yeast strain during its proline production, unless otherwisespecified. More specifically, to determine whether a yeast strain meetsthis requirement, a boiling water extract obtained from this targetyeast strain will be measured for its free amino acid composition. Inthis case, the target yeast strain provided for boiling water extractionhas been cultured under conditions effective for producing a specificfree amino acid (e.g., proline), as described herein. An example ofeffective conditions is shaking or spinner culturing for 10 hours orlonger using YPD medium (1.0% Bacto yeast extract, 2.0% Bacto peptone,2.0% glucose).

The yeast strain of the present invention may comprise free proline inan amount of 8.0% or more of the free amino acid composition, but it ispreferably a yeast strain which may comprise free proline in an amountof 15.0% or more of the free amino acid composition. Another example ofthe yeast strain of the present invention is a yeast strain belonging tothe genus Saccharomyces or Candida and having a tolerance toazetidine-2-carboxylic acid (AZC), wherein upon culturing underconditions effective for proline production and also effective forglycine and/or alanine production, a boiling water extract of theresulting cells may comprise free proline in an amount of 6.0% or moreof the free amino acid composition and free glycine in an amount of 5.0%or more of the free amino acid composition.

Examples of a “strain having a high proline-producing capacity and, ifdesired, having a high glycine-producing capacity and/or a highalanine-producing capacity” as used herein include: a yeast strainwherein upon culturing under conditions effective for prolineproduction, a boiling water extract of the resulting cells may comprisefree proline in an amount of 8.0% or more of the free amino acidcomposition (preferably a yeast strain which may comprise free prolinein an amount of 15.0% or more of the free amino acid composition); and ayeast strain wherein upon culturing under conditions effective forproline production and also effective for glycine and/or alanineproduction, a boiling water extract of the resulting cells may comprisefree proline in an amount of 6.0% or more of the free amino acidcomposition and free glycine in an amount of 5.0% or more of the freeamino acid composition.

The yeast strain of the present invention can be obtained by screeningstrains having a high proline-producing capacity, as described above.Microbiological properties of Saccharomyces cerevisiae JT-YE-P-52(Accession No. ABP-10725) obtained herein in the Example section, whichis an example of the yeast strain of the present invention, will beshown in the table below.

TABLE 1 Microbiological properties of JT-YE-P-52 Morphology Round tooval Size 3 to 7 μm Sporulation Presence Carbon source assimilation andfermentation Assimilation Fermentation D-Glucose + + D-Galactose + +Saccharose + + Maltose + + Lactose − − Raffinose + + Starch − −Morphology: Yeast cells were cultured in YPD medium and observed under amicroscope. Size: Yeast cells were cultured in YPD medium and observedunder a microscope, as in the case of morphology. Sporulation: Yeastcells grown on YPD agar medium were inoculated onto Sharman agar medium,cultured at 20° C. to 25° C. for 3 to 10 days, and observed under amicroscope to confirm the presence or absence of sporulation.

Carbon source assimilation and fermentation: Assimilation was analyzedas follows. A loopful of fresh yeast cells grown on YPD agar medium wassuspended in 5 ml sterilized water, washed twice with sterilized waterby centrifugation and then suspended again in 5 ml sterilized water. Thesuspension thus obtained (0.1 ml) was inoculated into tubes (Sarstedttubes, 101 mm×16.5 mm) containing 5 ml sterilized medium supplementedwith various carbon sources, respectively (Yeast nitrogen base 0.67 g,various carbon sources 0.1 g each, water 10 ml), grown in shakingculture at 30° C. for 48 hours and then measured for absorbance at 660nm to determine cell growth by the degree of turbidity. To analyzefermentation, a yeast cell suspension prepared as described above (0.1ml) was inoculated into glass tubes (180 mm×15 mm) containing the samemedium (10 ml) and equipped with a Durham's tube, grown in staticculture at 30° C. for 1 week and then confirmed for the presence orabsence of air bubbles in the Durham's tube.

[Other Embodiments and Applications, Etc.]

The yeast extract and seasoning composition of the present invention(hereinafter referred to as “the yeast extract and others of the presentinvention”) may be in the form of liquid, paste, powder or granule, andmay also be used in combination with other seasonings or additivesacceptable as foods.

The yeast extract and others of the present invention can beincorporated into various foods (including health foods). In addition tofoods, they can be incorporated into other products such as medicaments,cosmetics and pet foods.

The yeast extract and others of the present invention have the samesweetness quality as hydrolyzed animal proteins, and hence can be usedas their alternatives in foods for which hydrolyzed animal proteins aresuitable.

The yeast extract and others of the present invention can be used inmeat, fish and vegetarian dishes. Specific examples of foods includewhite sauce, meat sauce, demiglace sauce, tomato sauce, curry, stew,cream soup, minestrone, sauces (e.g., Worcestershire sauce, middle-thicksauce, thick sauce, pork cutlet sauce, Okonomiyaki sauce (sauce forJapanese-style hotplate pizza), Yakisoba sauce (sauce for friednoodles), Takoyaki sauce (sauce for fried octopus dumplings)), seasoningsoy sauces (e.g., those for Chinese noodles, buckwheat noodles and whitewheat noodles), hot pot soup stock (e.g., Oden soup stock (stock forfish cake in soup)), miso soup, chop suey, Chinese-style donburi(stir-fried vegetables & seafood on rice), Chinese fried rice, Chinesefried dumplings, Chinese steamed dumplings, Chinese buns, junk foods,dressings, dried seasoning powder, dipping sauces (dipping sauce forgrilled meat), Tsukudani (foods boiled in soy sauce), etc.

The yeast extract and others of the present invention can be used infrozen foods, ready-to-eat foods and instant foods.

The amount for use in foods may be determined as appropriate by thoseskilled in the art. For example, the yeast extract and others of thepresent invention may be added in an amount of 0.01% to 1% per weight offood when eaten or drunk.

ADVANTAGES OF THE INVENTION

The present invention enables the provision of a seasoning being freefrom MCP and DCP and being rich in sweet amino acids, and also enablesthe impartation of a sweetness characteristic of hydrolyzed animalproteins to foods.

Moreover, the present invention allows the predominance of sweetness dueto the reduced content of amino acids generally known to be bitter,i.e., isoleucine, leucine and arginine.

The present invention will now be described in more detail by way of thefollowing examples.

Example 1

A bakers' yeast strain JT-1 (Japan Tobacco Inc., Japan) was cultured inYPD medium (1.0% Bacto yeast extract (DIFCO), 2.0% Bacto peptone(DIFCO), 2.0% glucose) until reaching the logarithmic growth phase, andthen suspended in a 0.067 M sodium phosphate solution, followed by UVirradiation for 2 minutes while stirring. The strain was then culturedat 32° C. for 5 days on a minimal medium (0.67% yeast nitrogen base w/oamino acid (DIFCO), 2.0% glucose, 2.0% agar) containing 10 mg/ml AZC(SIGMA) to obtain 147 AZC-tolerant strains. These colonies were eachcultured in 50 ml YPD medium for an additional 24 hours and thencentrifuged to collect the cells. The lyophilized cells were extractedwith boiling water at 95° C. for 20 minutes and analyzed for theirproline and glycine contents with an amino acid analyzer (Hitachi modelL-8900). Among candidate strains, JT-YE-P-52 was obtained as a strainrich in proline and glycine.

Example 2

The strain JT-YE-P-52 was cultured with shaking for 24 hours using 10baffled 500 ml Erlenmeyer flasks containing 200 ml YPD medium, and thencentrifuged at 5000 rpm for 10 minutes to obtain wet cells (48.1 g).After addition of water (70 ml), the cells were extracted at 95° C. for20 minutes while stirring. After extraction, centrifugation wasperformed at 5000 rpm for 10 minutes to separate extraction residues,thereby obtaining a yeast extract (yeast extract B, 76 g). This extractwas measured for its free amino acid composition per solid content toobtain the results as shown in Table 1. For comparison purposes, theamino acid composition of a control extract (yeast extract A) preparedfrom the unmutated JT-1 strain is shown. Compared to the unmutated case,alanine remained unchanged, while glycine and proline increased. Inaddition, the percentages of bitter amino acids leucine, isoleucine andarginine were reduced to about half.

TABLE 2 Free amino acid composition of the extract of the presentinvention Yeast extract A Yeast extract B Amino Free Amino Free acid(%)/ amino acid acid (%)/ amino acid solid content composition solidcontent composition Asp 0.076 0.32 0.144 0.54 Thr 0.964 4.04 1.564 5.85Ser 0.624 2.61 0.612 2.29 Glu 9.676 40.55 8.276 30.95 Gly 0.404 1.691.372 5.13 Ala 3.664 15.35 3.732 13.96 (Cys)2 0.576 2.41 1.588 5.94 Val1.024 4.29 0.868 3.25 Met 0.044 0.18 0.064 0.24 Ile 0.932 3.91 0.4761.78 Leu 1.024 4.29 0.708 2.65 Tyr 0.272 1.14 0.352 1.32 Phe 0.312 1.310.392 1.47 Lys 1.1 4.61 0.156 0.58 His 0.188 0.79 0.28 1.05 Arg 2.77611.63 1.556 5.82 Pro 0.208 0.87 4.596 17.19 Total 23.864 100.00 26.736100 Bitterness Bitterness 19.83 10.25

Example 3

To study taste effects for the extract of the present invention shown inExample 2, the following experiment was performed. First, 18 trainedpanelists were allowed to taste sugar, a hydrolyzed animal protein(Ekisutorahto; JT Foods Co., Ltd., Japan), yeast extract 21TF (JT FoodsCo., Ltd., Japan), yeast extract 21A (JT Foods Co., Ltd., Japan) and thecontrol extract from Example 1 (yeast extract A), whereby the panelistswere given to understand that there was a difference in the quality ofsweetness between sugar and the hydrolyzed animal protein, and were alsomade to recognize the sweetness quality of the hydrolyzed animalprotein. Likewise, the panelists were also made to recognize that thecontrol extract was free from the sweetness provided by the hydrolyzedanimal protein.

The yeast extract A, which had been prepared from the strain JT-1 (theoriginal parent strain used for mutation in the present invention) inthe same manner as shown in Example 2, was added with proline (Wako PureChemical Industries, Ltd., Japan) at a final content of 5.0%, 7.0%,8.0%, 9.0%, 13.0%, 14.0% or 15.0% of the amino acid composition, andthen adjusted with hot water to 1% solid content. Separately, the yeastextract A was directly used to prepare a 1% solid content solution inhot water. To adjust the salt concentration at which the ability todetect sweetness is affected, these samples were added with salt in afinal amount of 26% of the solid content, and then provided for asensory evaluation. In the sensory evaluation, test samples wereevaluated for sweetness intensity on a ten-point scale, assuming that asample having the same sweetness intensity as the hydrolyzed animalprotein “Ekisutorahto” scored 5 points. These samples were alsoevaluated for sweetness quality on a five-point scale, assuming that asample having the same sweetness quality as the hydrolyzed animalprotein scored 5 points, while a sample whose sweetness quality wasgreatly different from that of the hydrolyzed animal protein scored 1point. Mean scores were obtained for each sample. This test wasperformed by the same 18 trained panelists as shown above.

The results obtained are as shown in Table 2. The samples containing 8%or more proline showed an increase in their sweetness and had asweetness quality close to that of the hydrolyzed animal protein. Astrong sweetness was observed in the sample containing 15% proline, andits sweetness quality was closer to that of the hydrolyzed animalprotein. Moreover, the yeast extract B prepared in Example 2 showed astronger sweetness and had a sweetness quality much closer to that ofthe hydrolyzed animal protein. This would be because the percentages ofbitter amino acids were low, as shown in the table below.

TABLE 3 Result of a sensory evaluation of samples Sweetness SweetnessTest sample intensity quality Comment Yeast extract A 1.2 1.2 (prolinecomposition: 0.87%) Amino acid-supplemented 2.1 1.4 sample 1 (5%proline) Amino acid-supplemented 2.1 1.6 sample 2 (7% proline) Aminoacid-supplemented 3.4 3.7 This sample is of advance taste type, hassample 3 a clear and well-harmonized taste, and (8% proline) preventsany heavy flavor from remaining in the mouth. Its sweetness quality isclose to that characteristic of hydrolyzed animal proteins, rather thana heavy sweetness as observed in sugar. Amino acid-supplemented 3.4 3.7sample 4 (9% proline) Amino acid-supplemented 3.6 3.7 sample 5 (13%proline) Amino acid-supplemented 3.6 3.7 sample 6 (14% proline) Aminoacid-supplemented 4.4 4.5 This sample is of advance taste type, hassample 7 a clear and well-harmonized taste, and (15% proline) preventsany heavy flavor from remaining in the mouth. Its sweetness quality isvery close to that characteristic of hydrolyzed animal proteins, ratherthan a heavy sweetness as observed in sugar. Yeast extract B 4.8 4.9This sample is of advance taste type, has a clear and well-harmonizedtaste, and prevents any heavy flavor from remaining in the mouth. Itssweetness quality is the same as that characteristic of hydrolyzedanimal proteins, rather than a heavy sweetness as observed in sugar.Hydrolyzed animal protein 5 5

Example 4

To study taste effects for the extract of the present invention shown inExample 2, the following experiment was performed.

As shown in Table 3, the yeast extract A was supplemented with prolineat a final content of 6.0% and further with glycine at a final contentof 3.0%, 4.0% or 5.0% to prepare test samples. The same procedure asshown in Example 3 was repeated to evaluate the sweetness intensity andquality of these samples in comparison with the yeast extract A, theyeast extract B or the hydrolyzed animal protein. The results indicatedthat the sample containing 5.0% glycine and 6.0% or more proline showedan increase in its sweetness. Likewise, the same test was also performedon the yeast extract A supplemented with glycine at a final content of5.0% and further with proline at a final content of 3.0%, 4.0%, 5.0% or6.0%, indicating that the sample containing 6.0% proline provided asweetness quality close to that of the hydrolyzed animal protein. Itshould be noted that the organoleptic test was performed as described inExample 3.

TABLE 4 Result of a sensory evaluation of samples Sweetness SweetnessTest sample intensity quality Comment Yeast extract A (1.69% glycine,1.2 1.2 0.87% proline) Amino acid-supplemented sample 1 3 2.3 (3.0%glycine, 6.0% proline) Amino acid-supplemented sample 2 3.4 2.8 Thissample is of advance (4.0% glycine, 6.0% proline) taste type, has aclear and well-harmonized taste, and prevents any heavy flavor fromremaining in the mouth. Its sweetness quality is close to thatcharacteristic of hydrolyzed animal proteins, rather than a heavysweetness as observed in sugar. Amino acid-supplemented sample 3 4.5 4(5.0% glycine, 6.0% proline) Amino acid-supplemented sample 4 3.2 2.8(5.0% glycine, 3.0% proline Amino acid-supplemented sample 5 3.3 3.2(5.0% glycine, 4.0% proline) Amino acid-supplemented sample 6 4 3.3 Thissample is of advance (5.0% glycine, 5.0% proline) taste type, has aclear and well-harmonized taste, and prevents any heavy flavor fromremaining in the mouth. Its sweetness quality is close to thatcharacteristic of hydrolyzed animal proteins, rather than a heavysweetness as observed in sugar. Yeast extract B 4.8 4.9 This sample isof advance taste type, has a clear and well-harmonized taste, andprevents any heavy flavor from remaining in the mouth. Its sweetnessquality is very close to that characteristic of hydrolyzed animalproteins, rather than a heavy sweetness as observed in sugar. Hydrolyzedanimal protein 5 5

Example 5

The extract of the present invention shown in Example 2, an extractprepared from the unmutated yeast cells, and a hydrolyzed animal proteinwere each lyophilized in a freeze-dryer and adjusted to give a saltconcentration of 26% per solid content. According to the recipe shown inTable 4, white sauces were prepared using these samples and compared fortheir sweetness intensity and quality. The results indicated that thewhite sauce prepared using the extract of the present invention provideda sweetness comparable to that of the hydrolyzed animal protein.

TABLE 5 Recipe of white sauce Milk 37.5 Onion 12.5 Bechamel 5 Carrot3.75 Short-necked clam (shelled) 3.75 Salad oil 0.5 Processed starch 0.5Salt 0.4 Consomme 0.1 Extract of the present invention 0.1 White pepper0.02 Garlic pepper 0.006 Water 35.88 Total 100.006

INDUSTRIAL APPLICABILITY

As described above, the present invention enables the production of aseasoning rich in sweet amino acids contained in hydrolyzed animalproteins without using hydrochloric acid hydrolysis techniques, andhence enables the provision of a MCP- and DCP-free seasoning toconsumers.

1-9. (canceled)
 10. A yeast extract comprising free proline in an amountof 8.0% or more of the free amino acid composition, and having ahydrolyzed animal protein-like sweetness.
 11. The yeast extractaccording to claim 10, which comprises free proline in an amount of15.0% or more of the free amino acid composition.
 12. A seasoningcomposition comprising a yeast extract, wherein the seasoningcomposition comprises free proline in an amount of 17.0% or more of thefree amino acid composition, free glycine in an amount of 5.0% or moreof the free anino acid composition, and free alanine in an amount of 12to 17% of the free amino acid composition.
 13. A yeast strain forpreparing the seasoning composition or the yeast extract according toany one of claims 10 to 12, which belongs to the genus Saccharomyces orCandida and having a tolerance to an analog of proline, and wherein uponculturing under conditions effective for proline production, a boilingwater extract of the resulting cells may comprise free proline in anamount of 8.0% or more of the free amino acid composition.
 14. The yeaststrain according to claim 13, which is Saccharomyces cerevisiaeJT-YE-P-52 (FERM BP-10725) or a mutant strain thereof having thefollowing microbiological properties: Morphology Round to oval Size 3 to7 μm Sporulation Presence Carbon source assimilation and fermentationAssimilation Fermentation D-Glucose + + D-Galactose + + Saccharose + +Maltose + + Lactose − − Raffinose + + Starch − −


15. The yeast extract according to claim 11, which comprises freeglycine in an amount of 5.0% or more of the free amino acid composition.16. The yeast extract according to claim 15, which comprises freealanine in an amount of 12 to 17% of the free amino acid composition.17. An agent for adding hydrolyzed animal protein-like sweetness to afood, wherein the agent comprises a yeast extract, and free proline inan amount of 6.0% or more of the free amino acid composition and freeglycine in an amount of 5.0% or more of the free amino acid composition.18. A method for adding hydrolyzed animal protein-like sweetness to afood, wherein the method comprises adding a composition comprising ayeast extract, and free proline in an amount of 6.0% or more of the fieeamino acid composition and free glycine in an amount of 5.0% or more ofthe free amino acid composition to the food.
 19. Use of a compositioncomprising a yeast extract, and free proline in an amount of 6.0% ormore of the free ainino acid composition and firee glycine in an amountof 5.0% or more of the free amino acid composition in the manufacture ofan agent for adding hydrolyzed animal protein-like sweetness to thefood.